10.1007%2Fs002170100373

7/27/2019 10.1007%2Fs002170100373

http://slidepdf.com/reader/full/1010072fs002170100373 1/5

Abstract An HPLC method for the determination of po-lar compounds in used frying oils/fats is described. It isbased on normal phase liquid chromatography, i.e., the

same separation mode as the reference method. The useof a mass-sensitive evaporative light scattering detector(ELSD) permits the quantification of the polar andnon-polar compounds under two symmetrical, baseline-resolved peaks. A polarity gradient is used for the elu-tion of the polar oil components. The results correlatewell with the official reference method (r2>0.97). Sim-plicity, speed, and low consumption of organic solventare the main advantages.

Keywords Polar compounds · Frying oils · Frying fats ·HPLC

Introduction

Frying oils/fats are used as a heat transfer medium forthe preparation of a variety of food products. Heating of such oils in the presence of oxygen, water, and foodcauses a gradual thermal degradation of the triglyce-rides. A large number of artifacts [1, 2, 3] have been re-ported.

Reference method for polar compounds

In order to monitor the quality of frying oils, a gravimet-ric column chromatography method was developed byGuhr and Waibel [4]. The measured polar compoundsare not a clearly defined group of substances, but areprobably best described as all substances which are re-tained in a silica column having been subjected to a de-

fined conditioning and elution process [4]. This methodhas been approved by international organizations likeIUPAC and AOAC as the official standard for the quality

control of frying fats. It is considered to be time-con-suming, labor-intensive [5], and reliability is not fullysatisfactory. A recent inter-laboratory test with 35 partic-ipating laboratories produced coefficients of variation(CV) of 17.2% (3.6% after the removal of eight outlyinglaboratories) [6].

Alternative methods

As an alternative, a number of chemical quick tests areavailable [7]. Acceptable correlations have been reportedbetween the polar compounds and the dielectric constant

as measured by the Food Oil Sensor (FOS) [5, 8]. NIR incombination with chemometric calibration techniques isreported to permit very fast measurements [9]. A “micromethod” described the potentials of miniaturization of the reference method [10].

The imitation of the reference method by other meth-odology is difficult, because of the vague definition of polar compounds. Most quick chemical tests as well asthe dielectric constant measurements are supposed to de-termine the concentration of polar moieties like carboxylgroups. However, aging of frying oils also producesoligomers, i.e., triglycerides linked by C-O-C or C-Cbonds. These as well as epoxides are retained on thesilica column together with the fraction of polar com-pounds, as shown by Aitzetmüller [11]. As a conse-quence, the determination of such a heterogeneous groupof compounds by means other than normal phase liquidchromatography is rather difficult.

There were attempts to imitate the reference methodby reversed phase liquid chromatography (RPLC) andrefractometry index (RI) detection [12]. We found it dif-ficult to obtain a clear-cut separation between the polarand non-polar fraction and noted a different selectivity.In RPLC the dimeric triglycerides are probably elutedwithin the non-polar fraction.

A. Kaufmann (✉) · B. Ryser · B. SuterOfficial Food Control Authority of the Canton of Zurich(Kantonales Labor Zürich), P.O. Box,CH-8030 Zürich, Switzerlande-mail: [email protected].: 0041-12525654, Fax: 0041-12624753

Eur Food Res Technol (2001) 213:372–376DOI 10.1007/s002170100373

O R I G I N A L PA P E R

Anton Kaufmann · Bianca Ryser · Bea Suter

HPLC with evaporative light scattering detection for the determination

of polar compounds in used frying oils

Received: 1 March 2001 / Published online: 15 August 2001© Springer-Verlag 2001

7/27/2019 10.1007%2Fs002170100373


373

Normal phase liquid chromatography

The most promising approach to substitute the referencemethod by an automated analysis deviates as little aspossible from the original principle. Aitzetmüller pro-posed frontal elution liquid chromatography [11] using amoving wire detector which is no longer commerciallyavailable. He not only reported one of the earliest at-

tempts to automate liquid chromatography (in 1973) butalso gave a still valid discussion about the unique char-acter of the normal phase liquid chromatography on sili-ca gels.

Chromatography on silica is based on both partition-ing and adsorption. A water-free silica surface containsactive sites and primarily separates by adsorption [13].If the activity of silica is reduced by water or ethanol,chromatography occurs by partitioning. As a conse-quence, the control of the water content of silica is es-sential. Silica adsorbs water from the eluent or desorbswater into a dry eluent until a steady state is reached.These changes cause drifting retention times [13]. If a

gradient is employed, a steady-state situation mightnever be reached. The concept of isohydric solvents wasintroduced [15, 16], permitting polarity gradients with-out affecting the water concentration of the stationaryphase. However, isohydric eluents require the careful ad- justment of the water concentration of the two eluents.Mostly a dry and a saturated solvent are mixed.

Detection technique

How can gravimetric quantification be replaced by achromatographic detection? UV response cannot be used

as it strongly differs for the compounds of interest. RIdetectors approach mass-sensitive measurement moreclosely, but can only be employed for isocratic runs.Hence the peak of the polar compounds (requiring gradi-ent elution) cannot be quantified. ELSD represents an-other mass-sensitive type of detector. Its main featuresare excellent gradient capability, ease of operation, andruggedness. However, because of the different underly-ing light scattering mechanisms, ELSDs do not producelinear response [14].

This paper presents an HPLC method which mimicsthe reference method for the determination of the polarcompounds [4] as closely as possible. The main advanta-

ges are speed, low labor-requirement, low solvent con-sumption, and improved repeatability. The validation of the method as well as the comparison with the referencemethod for polar compounds and the method measuringthe dielectric constant (FOS) will be presented in asecond paper [17].

Materials and methods

Instruments. The HPLC instrument consisted of a gradient pump(Gynkotek P 580A NDG/Agilent HP1100 quaternary), with an

autosampler (Gynkotek Gina 50), a column thermostat (Gynkotek STH 585), and a light scattering detector (Polymer LaboratoriesPL-ELS 1000).

The column flow rate was 2.0 ml/min, while the column temper-ature was held at 10 °C. Injection volume was 20 µl. The gradientprogram consisted of 0.0–0.5 min 0% B, 0.5–1.9 min 0–100%B,1.9–2.4 min 100% B, 2.4–2.9 min 100–0%B, 2.9-x min 0% B. Theconditioning time (x) required depends on the dead volume of theHPLC pump and might vary between 4 and 9 min. The dead vol-ume was determined by injecting the reference oil while maintain-

ing 100% eluent B. The conditioning time was increased stepwiseuntil the first peak of the reference oil (non-polar compounds) elut-ed between k ′ 0.6 and 1.0. Three consecutive injections were madefor any evaluated conditioning time in order to ensure that stableperformance was reached. A longer conditioning prolongs the k ′value of the first eluting peak while the k ′ value of the second peak (polar compounds) remains constant.

The ELSD spray temperature was set to 40 °C and the evapo-ration temperature to 90 °C. A nitrogen gas flow of 1 l/min wasused.

Chemicals and materials. Hexane 96% (multisolvent, HPLCgrade, Scharlau, Barcelona), ethanol, abs., HPLC quality, MächlerAG, Reinach, Switzerland), bidistilled water, diethyl ether (Ph.H.V.Siegfried, Zofingen, Switzerland). A reference oil (25–30% polarcompounds) was used as a standard.

A 30×4.6 mm i.d., 10 µm particle size cyano Nucleosil HPLCcolumn, (Macherey Nagel) was used for the separation. A peek tubing (3.0 m×0.5 mm i.d.) was installed between the column andthe detector.

Solutions. The extraction solvent consisted of hexane:diethylether, 10:3 (by volume), the mobile phase A of hexane, and mo-bile phase B of hexane:ethanol:water, 50:50:1 (by volume). Botheluents were stored in brown bottles.

Standards. Stock solution: 0.625±0.02 g reference oil warmed to40–50 °C was weighed in a graduated flask. The latter was filledwith extraction solvent to 50 ml. Reference oil was produced byprolonged heating of a frying fat in an open, air-exposed vesseluntil FOS measurement indicated values corresponding to 25–30%polar compounds. The exact concentration of polar compounds

was determined by the reference method (six independent determi-nations, each with an individually deactivated silica batch per-formed by three different operators).

Six standards solutions (A, B, C, D, E, F) were prepared bytransferring 1, 3, 6, 8, 10, and 12 ml stock solution into 50-mlgraduated flasks and filling up to the mark with hexane. In a re-frigerator solutions are stable for more than three months.

Preparation of samples. Warmed oil (15 drops, 40–50 °C) weredissolved in 20 ml extraction solvent. Then 0.4 ml of this solutionwas diluted in the HPLC sample vial with 1.2 ml hexane and thevial closed with a PTFE only or a Viton seal.

Calibration. Peak areas of the polar and non-polar compounds arecalibrated by a quadratic, non-zero offset calibration curve. Thecalibration included the concentration of the polar and non polar

compounds. For example, if 0.625 g reference oil of 30.4% polarcompound is used, standard A contains 0.174 g/l non-polar and0.076 g/l polar compounds. The percentage of polar compounds ina sample was calculated as polar compounds (%)=100×amountpolar/(amount non-polar+amount polar).

Results

Figure 1 shows a chromatogram of a heavily used (top)and a fresh frying oil. Two well separated peaks wereobtained in less than 2.5 min.

7/27/2019 10.1007%2Fs002170100373


374

Selection of the stationary and mobile phase

Separations on underivatized silica HPLC columns were

strongly affected by the water concentration in the elu-ent. A cyano-modified column used under normal phasechromatography conditions was preferred. Such phasesare equilibrated after a rather short period of time [13].

The reference method uses a mixture of petroleumether and diethyl ether as mobile phase. Because of thehigher point (to prevent cavitation) hexane and ethyl al-cohol were preferred. A column temperature of 10 °Cimproved the peak shape of the peak containing the non-polar compounds.

Conditioning/deactivation of the stationary phase

The selectivity of the column used by the referencemethod strongly depends on the water content of thesilica [10]. The cyano HPLC column used showed a sim-ilar behavior. After a gradient run it was deactivated andhad to be conditioned by hexane to restore the requiredactivity. Figure 2 shows the effect of varying the dura-tion of reconditioning on chromatography of a used fry-ing oil. The top chromatogram in Fig. 2 was obtained byinjection into 100% mobile phase B. The retention timeof the single peak (polar and apolar compounds) reflectsthe dead volume of the system.

The sample was injected into a column of drifting ac-tivity rather than stabilizing (equilibrating) the column at agiven activity. This speeds up the analysis and providesincreasing activity during chromatography of the non-po-lar compounds, causing an accentuated separation be-tween the non-polar and the polar compounds. Removalof the ethanol during conditioning increases column activ-ity, i.e., prolonged conditioning results in a more retentivecolumn. At the same time, more material of intermediatepolarity is shifted from the non-polar to the polar fraction.Selectivity of the separation must therefore be adjusted tothat of the reference method by reconditioning such thatthe same amount of polar material is found. Low dead

volume pumps like the tested Agilent 1100 dry the column

in such a short time that it is feasible to reach a stableequilibrium within a reasonable time. If such a pump isavailable, proper column activity can also be obtained byadding ethanol to the mobile phase A. Ethanol (0.1%) inthe mobile phase A permitted the elution of the non-polarcompounds within the specified k ′ range. Oils analyzed bythese two different approaches (conditioning vs equilibra-tion) did not produce significantly different results.

Ten frying oils from local restaurants were analyzedwith the reference method and by HPLC. HPLC analysiswas performed three times, each run with a different con-ditioning time. Figure 3 shows the correlation betweenthe classical polar compound and the HPLC method. Cor-relation was best at k ′ 0.5–1.0. The k ′ value and the peak width depends on the dead volume of the HPLC pumpmixing system. Hence reconditioning time has to be adjusted for a given pump in order to permit the elution of the non-polar peak at a k ′ value around 0.6–1.

Detection and calibration technique

Quantification required that the various compounds com-prised in the two peaks produce almost identical detectorresponses. Table 1 shows relative peak areas for an equal

Fig. 1 Chromatograms of a heavily used (top) and a fresh fryingoil

Fig. 2 Chromatograms of the same oil sample by identical gradi-ent ramps but variations of the conditioning time. The trace at thetop shows a chromatogram produced when injecting oil into 100%mobile phase B, hence reflecting the dead volume of the system.The following traces use 4, 5, and 6 min column conditioning be-fore starting the next injection/run. The LC pump used for this ex-periment was the Gynkotek P 580A

Table 1 Detector response for different substances: relative peak

area for equal amounts of different analytes

Analyte Absolute peak area

Sunflower oil 105Tristearate 1141,3-Dipalmitate 891,2-Dipalmitate 107Monopalmitate 106Epoxylates soy oil 93Palmitinic acid 86

7/27/2019 10.1007%2Fs002170100373


aration power of a short column with coarse particle di-ameter was still excessive, peak broadening was deliber-ately introduced by inserting a 3 m×0.5 mm i.d. tubingbetween the column and the detector. The strong peak-broadening introduced by this feature rendered negligi-ble the rather slight peak-broadening caused by partialseparation of analytes within the non-polar peak. Thereis no corresponding problem for the peak of the polar

compounds because of the steep gradient.

Ruggedness of the method

A number of tests were performed in order to assess therobustness of the method. The water content of thehexane (eluent A) and the composition of eluent B werevaried, with hardly any effect on the analytical re-sults. However, commercial hexane contains “polar com-pounds” which are concentrated on the column and elut-ed as a peak with the same retention time as the polarcompounds of the oil. Although this is accounted for by

the calibration curve (intercept), such a peak should bekept as small as possible. Presence of this contaminationdid not seem to be related to the advertised quality orprice of the hexane. As photoinduced reactions might beinvolved, eluents were be stored in brown eluent bottles.

Repeated injection from the same sample vial causedsepta material (silicone or rubber) to be partially dis-solved, which also generated a peak eluted at the reten-tion time of the polar compounds to be analyzed. HencePTFE only or Viton seals were used.

Discussion

The advantage of the proposed technique is the tight con-trol of a number of variance-generating factors (columnpacking, flow rate and column temperature). Further, a setof standards compensates for possibly deviating condi-tions, while the reference method relies on the assumptionthat all the non-polar compounds elute quantitatively withthe defined 150 ml eluent. The adjustable duration of con-ditioning permits a fine tuning of the method. The reten-tion time of the non-polar compounds is a quality relevantinformation, since it indicates the correctness of the col-umn deactivation for each chromatogram. Much HPLCsoftware permits the direct calculation of the polar com-pounds, hence producing, together with the chromato-gram, easily interpretable reports. However, the main ben-efits of the HPLC method over the classical method arethe simplicity and speed as well as the significant reduc-tion of labor and organic solvent cost.

References

1. Aitzetmüller K (1972) Fette Seifen Anstrichm 10:598–6022 Billek G (1973) Fette Seifen Anstrichf 75:582–5863. Al-Ismail K, Caboni MF (1998) Riv Ital Sostanze Grasse

75:235–239

375

amount of given analyte. Responses vary relatively little.Furthermore, it can be expected that different types of oils undergo similar thermal and hydrolytic decomposi-tion, such that the differences in detector response can beassumed to have no significant effect on the analysis.

There were suspicions that triglycerides could be de-tected at varying sensitivities, depending on whetherthey pass the detector cell as droplets of liquid or solidparticles. Even a strong reduction of the ELSD tempera-ture did not significantly affect peak areas. This was ob-served for oils as well as pure, crystallization-prone tri-stearate. Therefore, the response of an ELSD is a goodapproximation of the gravimetric reference method.

ELSDs produce an exponential, slightly sigmoid cali-bration curve [14]. The non-linearity of the detector isresponsible for a rather insidious effect. The non-polarcompounds, primarily intact triglycerides, are slightlyseparated, producing an asymmetric peak, whose shapedepends on the composition of the oil or fat. Peaks are,therefore, of a varying area/height ratio. Integration of such peaks is biased by a systematic error if the detectorshows non-linear response. For this reason the separationof the non-polar analytes was suppressed. Since the sep-

Fig. 3 The dead volume of an HPLC pump affects the condition-ing time. Hence this time has to be adjusted for every pump. Highcorrelation is observed if the k ′ value of the peak containing thenon-polar compounds is around 0.6–1

7/27/2019 10.1007%2Fs002170100373


10. Schulte E. (2000) Eur J Lipid Sci Technol 102:574–57911. Aitzetmüller K (1973) J Chromatogr 79:229–33412. Hein M, Isengard H (1997) Chromatographia 45:373–37713. Katz E (1998) Handbook of HPLC. Chromatographic science,

vol 78. Markus Dekker, New York 14. Onken J, Berger RG (1998) Dtsche Lebensm Runds 94:287–

29215. Thomas JP, Brun A (1977) J Chromatogr 139:21–4316. Thomas JP, Brun A (1979) J Chromatogr 172:107–13017. Kaufmann A, Ryser B (2001) Eur Food Res Technol (in press)

376

4. Guhr G, Waibel J (1978) Fette Seifen Anstrichm 80:106–1135. Gertz C (2000) Eur J Lipid Sci Technol 102:566–5726. Deutschen Gesellschaft für Fettwissenschaft (2000) Labor-

vergleichs-Untersuchung der Deutschen Gesellschaft für Fett-wissenschaft e.V. 2000

7. Dobarganes MC, Marquez-Ruiz G (1998) Grasas Aceites 49:331–335

8. Smith LM, Clifford AJ (1986) J Am Oil Chem Soc 63:1017–1023

9. Kehraus S, Harder J (1999) Lebensmittelchemie 53:9–11

Documents

10.1007%2Fs002170100373